The present invention relates to compositions and methods for controlling intra-cellular pH, e.g., vacuolar pH in plant cells. In particular, it relates to the isolation and characterization of Ph genes and their use in controlling vacuolar pH.
The vacuole is an important component of plant cells and often accounts for the majority of the cell volume. The vacuole contains water and a variety of organic and inorganic compounds such as sugars, organic acids, proteins, anthocyanin pigments and excretory products such as calcium oxalate and tannin compounds.
The vacuole is surrounded by a membrane (tonoplast membrane) which regulates the movement of materials into and out of the vacuole. Thus, the plant cell controls the contents of the fluid or cell sap within the vacuole. One of the properties of the cell sap which is regulated by the plant cell is proton concentration or pH of the solution.
A number of proteins can affect vacuolar pH. For instance, vacuolar ATPases generate pH and electrical gradients by hydrolyzing ATP and pumping protons across the tonoplast membrane. Nelson et al., Trends in Biochemical Science, 14:113-116 (1989). Another enzyme that affects vacuolar pH is inorganic pyrophosphatase. In Arabidopsis, this enzyme is a single 81 kDa protein that is encoded by a single gene (Sarafian et al., Proc. Natl. Acad. Sci. USA 89:1775-1779 (1992).
A group of genes in plants, referred to as Ph genes, encode proteins that also affect vacuolar pH. In Petunia hybrida (petunia) a number of Ph genes are known to play a role in determining vacuolar pH, although none has been reported as isolated or sequenced. de Vlaming et al. Theor. Appl. Genet. 66:271-278 (1983) and Wiering, et al., in Monographs on Theoretical and Applied Genetics 9: Petunia, K. C. Sink, ed. (Springer-Verlag, Berlin 1984), pp. 49-67, Gerats et al., Dev. Gen. 10:561-568 (1989) all of which are incorporated herein by reference.
In yeast, at least 17 different vph (vacuolar pH genes) required for acidification have been identified. For example, the vph1 mutation causes an elevation of vacuolar pH. Preston et al., Proc. Natl. Acad. Sci. USA 86:7027-7031 (1989). This mutant has been shown to lack ATPase activity as a consequence of a lesion in a gene encoding a 95kDa integral membrane subunit of the ATPase. Manolson et al. J. Biol. Chem. 267:14294-14303 (1992). A second vph mutant, vat2, is defective in the synthesis of the 60 kDa subunit of ATPase and also exhibits an elevated vacuolar pH. Nelson et al. Proc. Natl. Acad. Sci. USA 87:3503-3507 (1990).
Vacuolar pH is important in determining a number of plant traits. For instance, in petunia, certain forms of anthocyanins have been shown to appear red at low pH (e.g., pH 5.5) and blue at higher pH values (e.g., pH 5.9). Timberlake et al., in The Flavonoids, Harborne, et al. eds. (Academic Press, New York, 1975) pp. 214-266. Thus, flower color has been shown to change as the pH of the corolla cell vacuole is altered. In addition, the acidity of fruits such as tomatoes or citrus fruits depends upon the pH of the vacuolar contents. Other traits known to be affected by vacuolar pH include seed coat development, female fertility, protein transport.
There is currently a need for methods of producing new plant varieties with modified traits affected by vacuolar pH, such as flower color and fruit acidity. Control of the expression of genes encoding proteins affecting vacuolar pH provides a useful approach to this problem. The present invention addresses these and other needs.